Anti-white spot syndrome virus (WSSV) autophagy associated gene Cq-Atg8 and preparation method and application thereof

A technology of cq-atg8 and gene, applied in the field of antiviral activity gene, can solve the problem that no effective drug for WSSV infection has been found yet, and achieve the effect of strong anti-WSSV activity

Active Publication Date: 2014-10-08
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Unfortunately, people have not yet found

Method used

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  • Anti-white spot syndrome virus (WSSV) autophagy associated gene Cq-Atg8 and preparation method and application thereof
  • Anti-white spot syndrome virus (WSSV) autophagy associated gene Cq-Atg8 and preparation method and application thereof
  • Anti-white spot syndrome virus (WSSV) autophagy associated gene Cq-Atg8 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Prokaryotic Recombination-Induced Expression and Affinity Purification of Red Claw Crawfish Cq-Atg8

[0028] According to the multiple cloning site of the pGEX4T-2 vector, the open reading frame sequence of Cq-Atg8 gene of red claw crayfish was cloned into the pGEX4T-2 vector, and the pGEX4T-2 / Cq-Atg8 recombinant expression vector was constructed, which can be expressed in the recombinant strain E. coli BL21 induces expression to produce rCq-Atg8.

[0029] induced expression

[0030] 1. Transform the constructed recombinant expression vector into E.coli BL21, and induce expression with IPTG.

[0031] 2. Pick a monoclonal colony and inoculate it in 5ml containing Amp + LB medium, 37°C, 200rpm shaker culture to OD 600 0.6 to 2.0.

[0032] 3. Inoculate 20ml containing Amp at a ratio of 1:100 + LB medium, 37°C, 200rpm shaker culture to OD 600 0.1 to 0.2.

[0033] 4. Add IPTG to a final concentration of 0.1 mM, and induce at 200 rpm at 28°C for a certain per...

Embodiment 2

[0045] Example 2 Recombinant expression rCq-Atg8 anti-WSSV experiment

[0046] Anti-WSSV activity identification of rCq-Atg8: take 4 μg rCq-Atg8, mix with WSSV; after incubation for 30 minutes, add it to the cultured crayfish hematopoietic stem cells, and mix; incubate for 6 hours, collect the cells in the culture well, and extract the total RNA of Hpt cells , Total RNA was reverse-transcribed into cDNA after 1 μg DNase I treatment.

[0047] Real-time PCR was used to detect the transcription and expression of WSSV virus genes IE1 and VP28 in the above samples, and the 16srRNA gene was used as an internal reference. The primers used are shown in Table 1.

[0048] Table 1

[0049]

[0050] The reaction system is as follows:

[0051]

[0052] Reaction conditions: polymerase activation at 95°C for 10 minutes; denaturation at 95°C for 15 seconds, annealing at 60°C for 1 minute, and 40 cycles.

[0053] Data analysis: use 7500system SDS software version1.3.1.21 software to a...

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Abstract

The invention relates to an anti-white spot syndrome virus (WSSV) autophagy associated gene Cq-Atg8, and a preparation method and application thereof. The anti-WSSV autophagy associated gene Cq-Atg8 is from C. quadricarinatus and is named Cq-Atg8. The preparation method comprises the steps of (1) constructing a Cq-Atg8 pronucleus recombinant expression vector; (2) leading the Cq-Atg8 pronucleus recombinant expression vector obtained in the step (1) into a host cell, and carrying out inducible expression on the host cell by using isopropyl-betad-thiogalactoside (IPTG), so as to obtain an expression product; and (3) separating and purifying the expression product obtained in the step (2) so as to obtain the recombinant protein (rCq-Atg8). The rCq-Atg8 can inhibit infection and duplication of WSSV remarkably, and therefore the rCq-Atg8 can be used in preparation of anti-WSSV medicines and anti-disease feed additives for animals.

Description

technical field [0001] The invention relates to an autophagy-related gene, in particular to an antiviral active gene isolated from Cherax quadricarinatus: Cherax quadricarinatus autophagy-related gene—Cq-Atg8. Background technique [0002] White spot syndrome virus (WSSV) is the main pathogen of prawns, and can also infect crayfish, crabs, and lobsters. This virus has caused great harm to many important aquatic crustacean economic farming. Unfortunately, people have not yet found an effective drug against WSSV infection. [0003] Autophagy-related gene (Autophagy-related gene, Atg) 8—Atg8 is a ubiquitin-like protein, which is an essential component of autophagosome membrane formation, elongation and fusion with lysosomes. In addition, through the interaction with adapters, Atg8 is also involved in the selective recognition of cargo in the cytoplasm and its transport to lysosomes for degradation (Weidberg H, Shvets E, Elazar Z. Biogenesis and cargo selectivity of autophagos...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435C12N15/70A61K38/17A61P31/20A23K1/17A23K1/18A23K50/80
Inventor 刘海鹏陈荣元王克坚
Owner XIAMEN UNIV
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